Production of glomus intraradices propagules, an arbuscular mycorrhizal fungus, in an airlift bioreactor

This work addresses the symbiotic culture of the arbuscular mycorrhizal (AM) fungus Glomus intraradices with Daucus carota hairy roots transformed by Agrobacterium rhizogenes, in two submerged culture systems: Petri dish and airlift bioreactor. AM fungi play an active role in plant nutrition and protection against plant pathogens. These fungi are obligate biotrophs as they depend on a host plant for their needs in carbohydrates. The effect of the mycorrhizal roots inoculum-to-medium volume ratio on the growth of both symbionts was studied. A critical inoculating condition was observed at approximately 0.6 g dry biomass (DW). L-1 medium, above which root growth was significantly reduced when using a low-salt minimal (M) liquid medium previously developed for hairy root-AM fungi co-culture. Below critical inoculum conditions the maximum specific root growth and specific G. intraradices spore production rates of 0.021 and 0.035 d-1, respectively, were observed for Petri dish cultures. Maximum spore production in the airlift bioreactor was ten times lower than that of Petri dish cultures and obtained with the lowest inoculum assessed (0.13 g DW. L-1 medium) with 1.82 x 10(5) +/- 4.05 x 10(4) (SEM) spores (g DW inoculum)-1 (L medium)-1 in 107 d. This work proposes a second-generation bioprocess for AM fungi propagule production in bioreactors. Copyright 1999 John Wiley & Sons, Inc.     

In Situ Multiple Sampling of Attached Bacteria for Scanning and Transmission Electron Microscopy

An in situ electron microscope sampling technique for characterizing cells attached to smooth surfaces is demonstrated with an ultraviolet-induced mutant of Streptococcus mutans. The sterilized sampling unit consists of a 9 cm plastic Petri dish containing a glass slide, a 12 mm round coverglass, and a coverglass with Formvar-carbon coated copper grids. After the bacterial culture in a liquid medium is incubated in the Petri dish, the slide with attached bacteria is washed in double-distilled water, air-dried, coated with platinum and carbon, and processed for replicas and shadowed specimens for transmission electron microscopy. The coverglass is similarly washed, fixed in 2% glutaraldehyde, air- or freeze-dried, coated with palladium/gold, and examined in the scanning electron microscope. The coverglass with grids is rinsed in double distilled water, the grids are transferred to a filter paper and stained with a loopful of 2% phosphotungstic acid at pH 5.5. The bacteria growing on the surface of the plastic Petri dish are fixed, dehydrated, and embedded in situ with Epon. Sectioned and stained specimens are then examined in the transmission electron microscope. This procedure also appears useful with such other attached systems as normal or infected tissue culture cells.     

A BACTERIAL COLONY ILLUMINATOR

An apparatus is described in which side illumination from strip lights gives clear illumination without glare of colonies against a black background. The Petri dish lies in an unrestricted field and removal of the lid is unnecessary.     

Competition and co-existence of Zoophthora radicans and Pandora blunckii, two co-occurring fungal pathogens of the diamondback moth, Plutella xylostella

The entomopathogenic fungi Zoophthora radicans and Pandora blunckii co-occur in field populations of Plutella xylostella and, therefore, are likely to interact during the infection process. We have investigated the possible outcomes of these interactions in the laboratory. Using four isolates, two of each fungal species, inter-specific interaction experiments were done in Petri dishes and on intact plants. In Petri dish experiments, larvae were inoculated directly using sporulating mats of mycelium, both species had the same opportunity to infect and only the relative concentration of conidia of each pathogen species applied was manipulated. In the intact plant experiments, larvae were placed onto fungus-contaminated plants, inoculation was passive and the probability of infection by either or both species of fungi depended on larval activity and proximity to inoculum. In the Petri dish experiment, the species with the largest concentration of conidia out-competed the other regardless of virulence, and results were similar in the intact plant experiment. The ecological implications for competition or co-existence of these two pathogens in the field are discussed.     

Surface colony growth in a controlled nutrient environment. 1 The exponential law.

An apparatus designed to study the growth rates of surface colonies in constant conditions, i.e. not affected by nutrient diffusion as in a closed Petri dish, is described. In contrast to classical experiments in closed systems, an exponential growth of colony radius is obtained for a period of more than 72 h. Nutrient concentration gradients are shown to be eliminated by analytical techniques.     

Numerical modeling and dosimetry of the 35 mm Petri dish under 46 GHz millimeter wave exposure

Experimental studies on effects of millimeter wave (MMW) exposure on cells cultured in Petri dishes have attracted interest in recent decades. To improve the quantification of the biological responses toward the MMW energy, an accurate and precise MMW dosimetry is to be provided. By using the finite difference time domain (FDTD) method, the numerical dosimetry is performed for a typical 35 mm Petri dish under 46 GHz continuous MMW exposure from an irradiator of a specified power pattern. With the aim of building a precise model, the meniscus at the interface between the culture solution and the Petri dish sidewall is taken into account, followed by the modeling of smooth edges of the Petri dish. The trilinear interpolation is introduced to assist the FDTD method to obtain a more precise dosimetric assessment. The specific absorption rate (SAR) distributions in the cornea cells covered by culture solution in the Petri dish are calculated and compared to display the effects of using Petri dish models of various precision and the trilinear interpolation on dosimetry results. In addition, the SAR distribution in the cells is analyzed to study its homogeneity. The results indicate that the precise Petri dish model and the application of the trilinear interpolation are helpful in improving the precision of numerical dosimetry. It is also revealed that the inhomogeneity of the SAR distribution is well beyond neglect, which deserves cautious consideration in experiments investigating MMW effects on cells in vitro.     

A simple method of isolation and purification of cultures of wood-rotting fungi

A simple method of isolation and purification of cultures of higher fungi, mainly wood-rotting fungi, without special requirements for the presence of nitrogencontaining nutrients is described. Parts of fruiting bodies, spores or infected wood is inoculated on Petri dishes with an agar medium of the following composition: 1.0 g KH2PO4, 0.2 g MgSO4 - 7 H2O, 0.1 g caSO4, 0.01 g Fe2(SO4)3, 10.0 g glucose, tap water to 1 litre; 20.0 g agar. This medium does not suit most of the contaminants but fungal hyphae overgrow the whole surface of the dish so that a purified culture can be obtained from parts distant from the inoculation site.     

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.Download video file.^{(24M, mov)}     

Flexible Sheet-Type Sensor for Noninvasive Measurement of Cellular Oxygen Metabolism on a Culture Dish

A novel flexible sensor was developed for the noninvasive oxygen metabolism measurement of cultivated cells and tissues. This device is composed of a transparent double-layered polymer sheet of ethylene-vinyl alcohol (EVOH) and poly(dimethylsiloxane) (PDMS) having an array of microhole structures of 90 μm diameter and 50 μm depth on its surface. All the microhole structures were equipped with a 1-μm-thick optical chemical sensing layer of platinum porphyrin-fluoropolymer on their bottom. The three-dimensional microstructures of the sensor were fabricated by a newly developed simple and low-cost production method named self-aligned hot embossing. The device was designed to be attached slightly above the cells cultivated on a dish to form a temporarily closed microspace over the target cells during measurement. Since the change in oxygen concentration is relatively fast in the microcompartmentalized culture medium, a rapid evaluation of the oxygen consumption rate is possible by measuring the phosphorescence lifetime of the platinum porphyrin-fluoropolymer. The combined use of the device and an automated optical measurement system enabled the high-throughput sensing of cellular oxygen consumption (100 points/min). We monitored the oxygen metabolism of the human breast cancer cell line MCF7 on a Petri dish and evaluated the oxygen consumption rate to be 0.72 ± 0.12 fmol/min/cell. Furthermore, to demonstrate the utility of the developed sensing system, we demonstrated the mapping of the oxygen consumption rate of rat brain slices and succeeded in visualizing a clear difference among the layer structures of the hippocampus, i.e., the cornu ammonis (CA1 and CA3) and dentate gyrus (DG).     

Biological Control of Phytopathogenic Fungi by Fatty Acids

The aim of the present study was to evaluate the antifungal activity of fatty acids against phytopathogenic fungi. Two pot experiments were conducted by mixing palmitic and oleic acids in the soil in which poor plant growth was observed. In addition, the antifungal activities of nine fatty acids (butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, oleic acid, and linoleic acid) against four phytopathogenic fungi: Alternaria solani, Colletotrichum lagenarium, Fusarium oxysporum f. sp. Cucumerinum, and Fusarium oxysporum f. sp. lycopersici, were assessed by measuring mycelial growth and spore germination via Petri dish assay. The results of the pot experiments showed that the mixture of palmitic and oleic acids enhanced the growth of the seedlings of continuous-tomato and continuous-cucumber. Except for oleic acid, in the Petri dish assay, the fatty acids tested were observed to inhibit the mycelial growth of one or more tested fungi. In addition to the suppression of mycelial growth, butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, and palmitic acid showed an inhibitory effect against spore germination and the extent of inhibition varied with both the type of fatty acids, and the fungi. In particular, capric acid displayed strong inhibitory effect against C. lagenarium on the mycelial growth and spore germination. The saturated fatty acids, i.e. palmitic acids, showed stronger antifungal activity than the unsaturated fatty acids, i.e. oleic acid. It suggests that fatty acids might be applicable to exploring for alternative approaches to integrated control of phytopathogens.     

Heavy metal accumulation in wheat and barley: The effects of soil presence and liquid manure amendment

An experiment was undertaken to evaluate the effect of liquid manure amendment on heavy metal accumulation in wheat and barley. For this purpose, both kinds of seedlings were grown simultaneously in a Petri dish, while wheat seedlings were also grown in pots containing unpolluted agricultural soil. All of the seedlings were irrigated with one of the three prepared solutions: artificial rainwater solution, heavy metal solution and liquid manure solution containing NH₄NO₃, H₃PO₄ and KOH along with equal amounts of heavy metals as in the second solution. Twenty days later, 1 g of plant tissue was digested with the mixture of HNO₃ and H₂O₂ for ICP-OES/HG-ICP-OES analysis. The results showed that the uptake of arsenic and mercury was highest for both plants grown in a Petri dish. Furthermore, the wheat grown in a Petri dish also had a high content of nickel, cadmium and copper, while the pot-grown wheat contained high amounts of iron and manganese, probably due to the adsorption of nickel, cadmium, copper and mercury on soil phases. The lower uptake of all heavy metals was observed after the amendment of liquid manure, with the exception of manganese in wheat and mercury in all plants.     

The effects of natural biofilms on the reattachment of young adult zebra mussels to artificial substrata

This laboratory study examined the effects of natural biofilms on the reattachment of young adult zebra mussels, Dreissena polymorpha, in Petri dishes. Natural biofilms were developed in glass and polystyrene Petri dishes using water samples collected at various times of the year. Biofilms were developed over 1, 3, 8, and 14 d. Controls were clean glass and polystyrene Petri dishes. Zebra mussels collected from the field (< or = 10 mm, ventral length) were placed in the dishes and their reattachment by byssal threads was recorded after 1 d. Zebra mussels reattached to the dish surface or the shells of other mussels in the dish, or remained unattached. The data indicate that reattachment to clean glass was greater than to clean polystyrene (p < or = 0.05, ANOVA), but there were no consistent differences between reattachment to filmed polystyrene and filmed glass dish surfaces. Zebra mussels in control and filmed glass dishes reattached in higher percentages to the dish surface compared to the shells of other mussels (p < or = 0.05, ANOVA). There was no difference in mussel of reattachment between the dish surface and the shells of other mussels in most control polystyrene dishes (p > 0.05, ANOVA), whereas in filmed polystyrene the percentage of reattachment to the dish surface was greater than to the shells of other mussels (p < or = 0.05, ANOVA). These results indicate that substratum wettability and the presence of biofilms on some types of substrata can be factors in the reattachment of young adult zebra mussels.     

Numerical and experimental dosimetry of petri dish exposure setups

Crawford TEM cells are often used to expose cell cultures or small animals in order to study the effects caused by high-frequency fields. They are self-contained, easy-to-use setups that provide a rather homogeneous field distribution in a large area around its center, corresponding approximately to far-field conditions. However, a number of conditions must be met if such TEM cells are intended to be used for in vitro experiments. For instance, poor interaction with the incident field must be maintained to avoid significant field disturbances in the TEM cell. This is best achieved with E-polarization, i.e., when the E-field vector is normal to the investigated cell layer lining the bottom of a synthetic Petri dish. In addition, E-polarization provides the most homogeneous field distribution of all polarizations within the entire layer of cells. In this paper, we present a detailed dosimetric assessment for 60 and 100 mm Petri dishes as well as for a 48-well titer plate at 835 MHz. The dosimetry was performed by using numerical computations. The modeling and the simplifications are validated by a second numerical technique and by experimental measurements. For thin liquid layers, an approximation formula is provided with which the induced field strength for many other experiments conducted in Petri dishes can be assessed reliably. © 1996 Wiley-Liss, Inc.     

Isolation and identification of root-inhibiting compounds from corn gluten hydrolysate

Interest has centered on the use of plantderived compounds as natural herbicides, and they are considered to represent an environmentally sound approach to weed control. Corn gluten hydrolysate, found to have a growth-regulating effect on the root system of germinating grass seeds, has been suggested as a natural herbicide. A protocol was developed to extract, isolate, and identify the root-inhibiting compounds from corn gluten hydrolysate aqueous solution and a perennial ryegrass (Lolium perenne L.). A Petri dish bioassay was used to test the root-inhibiting activity. Five bioactive dipeptides were isolated by using Sephadex G-15 gel filtration, solid-phase extraction, and C18 reversed-phase high-performance liquid chromatography procedures. The five dipeptides were glutaminyl-glutamine, alaninyl-asparagine, alaninyl-glutamine, glycinyl-alanine, and alaninyl-alanine. Their root-inhibiting activity on perennial ryegrass was demonstrated in Petri dish bioassays.Journal Paper No. J-15597 of Iowa Agriculture and Home Economics Experiment Station, Ames, IA Project No. 3149     

Method of determining the antibiotic sensitivity of anaerobic microorganisms using standard disks

Three variants of the procedure for determination of antibiotic sensitivity in anaerobic microorganisms with the use of standard paper discs were developed. According to the first variant the solid nutrient medium is melted at 46 degrees C and mixed with the culture of the microbe being tested. The mixture is added to the cover of a Petri dish. When the medium becomes solid, antibiotic sensitivity discs are placed onto the agar surface. After that one more layer of the medium is added. The medium is allowed to solidify and some more medium is poured near the cover edge. Immediately after that the Petri dish is placed with its flat surface onto the agar layer in its cover. According to the first and second variants the mixture of the medium and culture is added to a Petri dish and immediately a transparent gas-proof polymer film of the dish size is placed onto the agar surface. Previously antibiotic paper discs or solutions are fixed on the films. THe incubation temperature for all three variants is 37 degrees C. The procedure allows one to observe the culture growth and to obtain the results earlier than in case the culture is incubated in an aerostate. The procedure is simple and saves labor and time.     

Co-inoculation of Ceratocystis polonica and Heterobasidion annosum with callus of two Norway spruce clones with different in vivo susceptibility

We have studied how callus cultures from two clones of Norway spruce influence the growth of two pathogens, Ceratocystis polonica and Heterobasidion annosum, when co-cultivated in vitro. In field experiments, trees of clone 409 were susceptible to both fungi, whereas clone 589 was less affected. Callus was cultured on medium containing cytokinins (benzylaminopurine, kinetin) and with or without auxin (2,4-dichlorophenoxyacetic acid). For co-cultivation with fungus, one piece of callus was placed towards the edge of each Petri dish. One and 14 days after inoculation with callus the dishes were co-inoculated with the fungus. Both clones strongly stimulated the initial growth of both fungi. Clone 589 inhibited the growth of both fungi when the fungi were inoculated one day after the callus. When the callus was cultured on medium without auxin for 14 days before co-inoculation clone 589 strongly inhibited the growth of both fungi, whereas clone 409 inhibited H. annosum only. This revised version was published online in June 2006 with corrections to the Cover Date.     

A Novel Polystyrene Dish for the Production of Anaerobiosis

S ummary : The design and construction of a polystyrene dish to be used with alkaline pyrogallol for the production of anaerobic conditions is described. Paper mats are used to carry the reducing system, and the use of cellulose adhesive tape to effect an airtight seal is recommended.     

A simple method for reducing moisture condensation on Petri dish lids

Condensation on the lids of Petri dishes, used to culture plant tissues, can often obscure the view of the contents of the dish and interfere with data collection. Under the high humidity conditions that exist in the culture container, a small temperature drop causes water to condense on the inside lid and sides of the container. Mild condensation causes “fogging” while continual or repeated rounds of condensation result in the formation of water droplets. To control condensation in the standard plant tissue culture Petri dish, a simple method was developed whereby the lid of the culture dish was modified, to buffer the lid from temperature fluctuations. Polymer discs, which were the same diameter as the Petri dish lid, were either placed on the top of the lids of existing dishes or surface-sterilized and used in place of the lid. Polymer discs of varying thicknesses and type, and possessing different thermal conductivities, were evaluated for their abilities to reduce the rate of condensation formation. Petri dishes with modified lids were placed under reduced temperature conditions. Condensation, forming on the lids of the dishes was quantified over time using image analysis. Gray value determinations indicated that the thicker polymer discs with the lowest thermal conductivities provided the best protection against condensation. Placement of polymer discs on the top of Petri dishes is a relatively simple method that can be used to buffer the lid from small temperature changes and minimize condensation problems.     

Degassing‐assisted patterning of cell culture surfaces

We developed an alternative patterning technique which is capable of producing both topographic and biochemical features for cell culture studies. This technique is based on microaspiration induced with a degassed poly (dimethylsiloxane) (PDMS) mold. After degassing in a rough vacuum chamber and placed on a sample surface, liquid solution can be aspired through channels and cavities created in the PDMS mold. Depending on the composition of the solution and the associated drying or incubation processes, a variety of surface patterns can be produced without applying external pressure. For demonstration, we fabricated agarose gel microwells and biomolecule patterns either on a glass plate or in a cell culture Petri dish, both applicable for cell culture studies. Biotechnol. Bioeng. 2010. 105: 854–859. © 2009 Wiley Periodicals, Inc.     

The growth of fungi and Arabidopsis thaliana is influenced by bacterial volatiles

Dual culture systems, which only allowed volatiles to cross the boundary of a bipartite Petri dish, were used to investigate the effects of bacterial volatiles on the growth of 14 fungi and A. thaliana. The majority of tested combinations exhibited dramatic growth retardations of fungi and A. thaliana, indicating that volatiles can act as antibiotics. It therefore can be concluded that bacterial volatiles influence the growth conditions of organisms in a community and in a habitat.Key words: chemical communication, bacterial volatiles, fungi, A. thaliana, Pseudomonas trivialis, Serratia plymuthica, Staphylococcus epidermidis